Compositions and methods useful for the prevention of malaria and dengue virus transmission

ABSTRACT

The present invention relates to the fields of malaria and dengue virus. More specifically, the present invention provides compositions and methods useful for the treatment and prevention of malaria and dengue virus. In particular embodiments, a composition comprises mosquito nectar feed and Chromobacterium sp_Panamam (Csp_P).

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a 35 U.S.C. § 371 U.S. national entry ofInternational Application PCT/US2015/047321, having an internationalfiling date of Aug. 28, 2015, which claims the benefit of U.S.Provisional Application No. 62/042,856, filed Aug. 28, 2014, U.S.Provisional Application No. 62/052,524, filed Sep. 19, 2014, U.S.Provisional Application 62/185,005, filed Jun. 26, 2015, the content ofeach of the aforementioned applications is herein incorporated byreference in their entirety.

STATEMENT OF GOVERNMENTAL INTEREST

This invention was made with government support under grant no.AI061576, grant no. AI059492, grant no. AI078997, and grant no.AI080161, all of which were awarded by the National Institutes ofHealth. The government has certain rights in the invention.

FIELD OF THE INVENTION

The present invention relates to the fields of malaria and dengue virus.More specifically, the present invention provides compositions andmethods useful for the prevention of malaria and dengue transmission.

INCORPORATION-BY-REFERENCE OF MATERIAL SUBMITTED ELECTRONICALLY

This application contains a sequence listing. It has been submittedelectronically via EFS-Web as an ASCII text file entitled“P12694-04_ST25.txt.” The sequence listing is 7,980 bytes in size, andwas created on Aug. 27, 2015. It is hereby incorporated by reference inits entirety.

BACKGROUND OF THE INVENTION

Plasmodium and dengue virus, the causative agents of the twomost-devastating vector-borne diseases, malaria and dengue, aretransmitted by the two most important mosquito vectors, Anophelesgambiae and Aedes aegypti, respectively. The lack of vaccines andeffective drugs, along with insecticide resistance, has rendered thecontrol of these important pathogens cumbersome, and call for thedevelopment of novel disease transmission blocking strategies.

SUMMARY OF THE INVENTION

Plasmodium and dengue virus, the causative agents of the twomost-devastating vector-borne diseases, malaria and dengue, aretransmitted by the two most important mosquito vectors, Anophelesgambiae and Aedes aegypti, respectively. The present inventorsdiscovered that Chromobacterium sp_Panamam (Csp_P) can effectivelycolonize the midgut of An. gambiae and Ae. Aegypti mosquitoes whenintroduced through an artificial nectar meal. Csp_P exposure reduces thesurvival of both the larval and adult mosquito stages, and therebyrepresents a potent entomopathogenic agent. Because Csp_P blocksPlasmodium falciparum and dengue virus infection in the mosquito gut, italso represents a disease transmission blocking agent. Theentomopathogenic and anti-pathogen properties of Csp_P render it astrong candidate for malaria and dengue control strategies.

The entomopathogenic, in vivo anti-dengue and anti-Plasmodium propertiesof Csp_P make this bacterium a particularly strong candidate for use innovel control strategies for these two most important vector-bornediseases. In particular embodiments, Csp_P can be used in a diseasecontrol strategy based on the direct exposure of larval or adult stagemosquitoes to this bacterium, or the entomopathogenic and anti-pathogenagents (molecules) it produces. Exposure of larvae to Csp_P or itsproduced entomopathogenic extracts or purified molecules could beachieved through direction administration in the breeding water.Exposure of adult mosquitoes to Csp_P or its produced antipathogenextracts or purified molecules could be achieved through artificialnectar feeding.

Csp_P is the first identified bacterium that exerts broad-spectrumanti-pathogen activity against Plasmodium and dengue virus in theirrespective vectors, along with entomopathogenic activity against larvaland adult stages of An. gambiae and Ae. Aegypti. Csp_P is the firstbacterium that has been shown to mediate these diverse activitiesthrough secreted molecules.

Accordingly, in one aspect, the present invention provides compositionsuseful for the prevention of malaria and dengue virus transmission. Incertain embodiments, the compositions are useful as a generalmosquitocidal agent and/or a malaria and dengue transmission-blockingagent. In particular embodiments, a composition comprises mosquitonectar feed and Chromobacterium sp_Panamam (Csp_P). In a specificembodiment, Csp-P is comprises a biofilm. The biofilm can be fresh ordesiccated. In another embodiment, Csp-P is comprises a culture. In afurther embodiment, Csp_P comprises a supernatant. In yet anotherembodiment, Csp_P comprises a filtrate. In certain embodiments, Csp_Phas the 16s rDNA gene sequence of SEQ ID NO:1. In certain embodiments,the nectar feed comprises one or more of sucrose, dextrose and fructose.

The present invention also provides methods for controlling malaria anddengue virus transmission via mosquitoes comprising the step of applyinga composition described herein in an area where the mosquitoes are to becontrolled. In specific embodiments, the mosquitoes comprise Anophelesand/or Aedes mosquitoes. In more specific embodiments, the Anophelesmosquitoes comprise Anopheles gambiae mosquitoes. In other embodiments,the Aedes mosquitoes comprise Aedes aegypti mosquitoes.

In another specific embodiment, the present invention providescompositions comprising a biofilm, supernatant, filtrate or extract of abiologically pure culture of Chromobacerium sp. (Csp_P). In oneembodiment, the biologically pure culture of Csp_P has the 16S rDNA genesequence of SEQ ID NO:1.

In further embodiments, the present invention provides a method forcontrolling Anopheles and Aedes mosquitoes comprising applying in anarea where the mosquitoes are to be controlled a composition comprisingan effective insect control amount of a supernatant, filtrate or extractof a biologically pure culture of Csp_P. In a specific embodiment, thecomposition further comprises a sugar source. In more particularembodiments, the sugar comprises sucrose, dextrose and/or fructose. Incertain embodiments, the Csp_P is the bacteria having thecharacteristics of ATCC Designation No. PTA-121570.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Csp_P colonization of the mosquito midgut. All mosquitoes wereexposed to Csp_P via sugar meal. To introduce Csp_P via sugar meal,adults were allowed to feed for 24 h on 1.5% sucrose containing Csp_Pliquid culture at a final concentration of ˜10⁸ CFU/ml for An. gambiaeand ˜10⁶ (A, B) or 10¹⁰ (F) CFU/ml for Ae. aegypti. For antibiotictreated mosquitoes, the prevalence of Csp_P was measured in Ae. aegyptiand An. gambiae midguts at 3 days post-exposure (A). The number ofcolony forming units (CFUs) of Csp_P was also measured in the midguts of(B) Ae. aegypti and (C) An. gambiae 3 days after exposure to Csp_P.Experiments for antibiotic treated Ae. aegypti and An. gambiae werereplicated at least three times. Final sample sizes: nAe.aegypti/PBS=37; nAe. aegypti/Csp_P=37; nAn. gambiae/PBS=30; nAn.gambiae/Csp_P=17. For septic (i.e., non-antibiotic treated) mosquitoes,the prevalence and bacterial load of Csp_P was measured in An. gambiaemidguts at 1 and 2 days post exposure (D, E). Experiments for septic An.gambiae were replicated twice. Final sample sizes: nAn. gambiae/PBS=30;nAn. gambiae/Csp_P/Day 1=20; nAn. gambiae/Csp_P/Day 2=8. Prevalence ofCsp_P was measured in Ae. aegypti midguts at 1 and 3 days post exposure(F). Experiments for septic Ae. aegypti were replicated twice. Finalsample sizes: nAe. aegypti/Csp_P/Day 1=19; nAe. aegypti/Csp_P/Day 3=20.Horizontal lines indicate mean values. The following transformation wasapplied to all raw CFU data: y=log 10(x+1), where x=original CFU countand y=plotted data values.

FIG. 2. Csp_P exposure causes high mortality in adults and larvae. Csp_Pwas experimentally introduced into the adult midgut via either a sugarmeal (A-D) or blood meal (E, F), and mortality was observed over 5-8days. To introduce Csp_P via sugar meal, adults were allowed to feed for24 h on 1.5% sucrose containing Csp_P liquid culture at a finalconcentration of ˜10⁸ CFU/ml for An. gambiae and ˜10⁶ or 10¹⁰ CFU/ml forAe. aegypti. Csp_P ingestion significantly decreased survival insugar-fed aseptic (i.e., pre-treated with antibiotics) An. gambiae (A,p<0.0001) and Ae. aegypti (B, p<0.0001). Each experiment was replicatedthree times. Total sample sizes: (A) PBS=149; (A) Csp_P=146; (B) PBS=70;(B) Csp_P=70. Ingestion of Csp_P significantly decreased survival insugar-fed septic (i.e., not treated with antibiotics) An. gambiae (C,p<0.0001). In septic Ae. aegypti, survival was significantly decreasedafter feeding on a 10¹⁰ CFU/ml sugar meal (D, p<0.0001) but not afterfeeding on a 10⁶ CFU/ml sugar meal (D, p=0.08). Experiments in C and Dwere replicated twice. Total sample sizes: (C) PBS=95; (C) Csp_P=124;(D) PBS=185; (D) Csp10^6=223; (D) Csp10^ 10=226. To introduce Csp_P viablood meal, Csp_P liquid culture (˜10⁸ CFU/ml) was mixed 1:1 with humanblood/serum and fed to septic An. gambiae (E) and Ae. aegypti (F)adults. Experiments were replicated three times with total sample sizes:(E) PBS=59; (E) Csp_P=51; (F) PBS=37; (F) Csp_P=62. The effects of†Csp_P on larval mortality were also tested by placing 2- to 4-day-oldAn. gambiae (G) and Ae. aegypti (H) larvae in water containing Csp_P ata starting concentration of 10⁶ CFU/ml and monitoring survival over 5days. Experiments were replicated 2-3 times with final sample sizes: (G)PBS=80; (G) Csp_P=60; (H) PBS=100; (H) Csp_P=60. P values reported abovewere obtained by performing pairwise Log-Rank Tests between PBS andCsp_P treatments. Survival curves were fitted using the Kaplan-Meiermethod. Vertical tick-marks indicate censored samples; in C and Dmultiple individuals were dissected on each day to measure Csp_Pprevalence and bacterial load for FIG. 1.

FIG. 3. Csp_P reduces mosquitoes' susceptibility to malaria and dengueinfection. In (A) and (C), antibiotic-treated adults were allowed tofeed for 24 h on 1.5% sucrose containing Csp_P liquid culture at a finalconcentration of ˜10⁸ CFU/ml for An. gambiae (A) and ˜10⁶ CFU/ml for Ae.aegypti (C). After introduction of Csp_P via the sugar meal, An. gambiaemosquitoes were given a blood meal that contained P. falciparum, and Ae.aegypti mosquitoes were given a blood meal that contained dengue virus.In (B), Csp_P (10⁶ CFU/ml) was introduced concurrently with P.falciparum via blood meal through blood feeding of antibiotic treatedAn. gambiae. In all experiments, PBS was used as thenon-Csp_(—)P-exposed control. At 7 days after infection, midguts weredissected. Oocysts were counted in P. falciparum-infected An. gambiaefemales, and dengue virus titers were assayed in dengue-infected Ae.aegypti females by conducting standard plaque assays. Experiments wereinitiated using similar numbers of adult females in each treatment (A, Bstarting numbers=45-50/trtmt, C starting numbers=30-40/treatment). Allexperiments were replicated at least three times with final samplessizes: (A) PBS=67, (A) Csp_P=14, (B) PBS=43, (B) Csp_P=8, (C) PBS=68,(C) Csp_P=45. Differences between treatments were assessed byMann-Whitney test (*, p<0.05; ***, p<0.001).

FIG. 4. Csp_P elicits immune gene expression in the mosquito. Inductionof the Cec1 promoter in the SUA-5B cell-line exposed to P. putida andChromobacterium sp_Panamam Csp_P. SUA5B cells expressing a luciferasereporter gene driven by a Cec1 promoter were exposed to increasingconcentrations of Csp_P and P. putida bacteria. Differences betweenbacteria treated samples and PBS control samples were assessed byDunnett's Multiple Comparison Test (**, p<0.01; ***, p<0.001).

FIG. 5. Csp_P has anti-Plasmodium and anti-dengue activity in vitro.Csp_P was grown under planktonic and/or biofilm conditions and testedfor anti-pathogen activity independent of the mosquito. Five differentpreparations of Csp_P were tested: (a) planktonic state liquid culture,(b) biofilm supernatant, (c) fresh biofilm, (d) desiccated biofilm, and(e) heat inactivated biofilm. (A) Csp_P 36-h biofilm has anti-parasiteactivity against asexual-stage P. falciparum. Csp_P cultures werefiltered using a 0.2-μm filter and mixed with ring-stage P. falciparumparasite cultures. SYBR green I was then added to each sample, andinhibition of asexual-stage P. falciparum by Csp_P was measured byassaying fluorescence relative to the negative control (parasite medium,standardized to 0% inhibition). Chloroquine was used as a positivecontrol and standardized to 100% inhibition. We performed a Tukey's teston the raw data to determine whether each bacterial treatment differedsignificantly from the PBS+LB control (*** p<0.001). (B) Csp_P hasanti-parasite activity against ookinete-stage P. falciparum. Csp_Pbacterial preparations were filtered using a 0.2-μm filter and mixedwith blood taken from female Swiss Webster mice infected with Renillaluciferase-expressing transgenic P. berghei. Ookinete-stage P. bergheiparasite counts were determined using the Renilla luciferase assaysystem, and percent inhibition by Csp_P was calculated relative to thenegative control (PBS+LB control, standardized to 0% inhibition). Weperformed a Tukey's test to determine whether each bacterial treatmentdiffered significantly from the control (*p<0.05, ***, p<0.001). (C)Csp_P 42-h biofilm has anti-parasite activity against gametocyte-stageP. falciparum. Csp_P cultures were filtered using a 0.2-μm filter andmixed with gametocyte-stage P. falciparum cultures. Erythrocytes wereexamined for gametocytes using Giemsa-stained blood films collected 3days after Csp_P exposure. The red X indicates that the supernatantcaused hemolysis and was therefore unusable. We determined gametocytedensity per 1000 RBCs for each sample and performed a Tukey's test todetermine whether each bacterial treatment significantly differed fromthe PBS+LB control (*p<0.05, *** p<0.001). (D) Csp_P has antidengueactivity. Each Csp_P bacterial preparation (75 μl, unfiltered) was mixedwith 75 μl MEM containing dengue virus serotype 2 and incubated at roomtemperature for 45 min. Samples were then filtered through a 0.2-μmfilter and used to infect BHK21-15 cells. Percent inhibition wascalculated as the percent decrease in PFU/ml relative to the negativecontrol (PBS+LB, standardized to 0% inhibition). We analyzed thesignificance of pairwise comparisons between each treatment and thecontrol using a Tukey's test (***, p<0.001). (E) Csp_P has anti-dengueactivity when virus is suspended in human blood. Biofilms from multiplebacteria were tested for anti-dengue activity. All bacteria tested wereisolated from field-caught Ae. aegypti mosquitoes. The biofilm from eachspecies was grown for 48 h at room temperature, and dengue virus mixed1:1 with human blood was added directly to the biofilm. After a 45-minincubation, the virus+blood/bilofilm solution was filtered and used toinfect C6/36 cells. Biofilm sup=biofilm supernatant, H. I. biofilm=heatinactivated biofilm, dess. biofilm=desiccated biofilm resuspended in1×PBS.

FIG. 6. Csp_P has anti-bacterial activity against many species commonlyfound in the midguts of Aedes and Anopheles mosquitoes. Csp_P wasstreaked on LB agar along with multiple bacterial species, and plateswere observed for formation of zones of inhibition around Csp_P.Ps.sp=Presudomonas sp., Pr.sp=Proteus sp., Cs.p_P=C.sp_P, C.viol=C.violaceum, Pa.sp=Paenobacillus sp., Co.sp=Comamonas sp.,Ac.sp=Acinetobacter sp., Ps.pu=Pseudomonas putida, En.sp=Enterobactersp., Pn.sp=Pantoea sp., Ps.sp=Pseudomonas sp., S.sp=Serratia sp.,Ch.sp=Chryseobacterium sp.

FIG. 7. Csp_P elicits immune gene expression in the mosquito midgut.Changes in the abundance of immune effector gene transcripts in themidgut of (A) Ae. aegypti and (B) An. gambiae mosquitoes were measuredafter the introduction of Csp_P via a sugar meal. For each gene, PBScontrols were standardized to a value of 1.0, and Csp_P-induced changesin gene expression are shown as -fold change above or below PBS-fedcontrols. CecG=cecropin G, DefC=defensin C, LysC=lysozyme C,CecE=cecropin E, Cec1=cecropin 1, Def1=defensin 1, PGRP-LC=peptidoglycanrecognition receptor LC, Rel2=Relish-like NF-κB transcription factor 2,Tep1=thioester protein 1, LRRD7=leucine-rich repeat domain protein 7(a.k.a., APL2 and LRIM17), FBN9=fibronectin 9. Mann Whitney Testscomparing deltaCT values between bacteria-fed and PBS-fed mosquitoes foreach gene were performed to determine significance (*, p<0.05).

FIG. 8. Effect of 36-h biofilm on gametocyte-stage P. falciparum. Csp_Pcultures were filtered using a 0.2-μm filter and mixed withgametocyte-stage P. falciparum cultures. Erythrocytes were examined forgametocytes using Giemsa-stained blood films collected 3 days afterCsp_P exposure. We determined gametocyte density per 1000 RBCs for eachsample and performed a Tukey's test to determine whether each bacterialtreatment significantly differed from the PBS+LB control. No treatmentswere significant, but biofilm 36-h supernatant trended towardsignificance (p=0.06).

FIG. 9. (A) Anti-dengue activity of fresh Csp_P biofilm is only weaklypresent after 24 h of growth at room temperature and becomes highlypotent after 48 h of growth. Dengue virus was mixed 1:1 with human bloodand directly exposed to Csp_P biofilm grown for 24 or 48 h. Samples wereincubated for 45 min and then collected, filtered, and used to infectC6/36 cells. (B) Dengue virus particles are not sequestered by Csp_Pbiofilm. We mixed dengue virus with Csp_P biofilm and incubated themixture for 45 min. We then centrifuged samples and used qRT-PCR toquantify viral RNA in the supernatant of the experimental (biofilm+DENV)and control (LB+DENV) treatments.

FIG. 10. (A) Assessing changes in pH caused by Csp_P biofilm. We exposeddengue virus to Csp_P biofilm, incubated for 45 min, and measured the pHof the medium. (B) Assessing the effect of pH on dengue virusinfectivity. We experimentally adjusted the pH of the MEM medium usingNaOH and HCl to values of 5.0, 7.7, 8.5, and 10.0. We mixed thepH-adjusted media with dengue virus-laden human blood and incubated for45 min., then collected and filtered the virus and used it to infectC6/36 cells.

FIG. 11. Crude biofilm extract does not have cytotoxic effects on insector mammalian cells. We used trypan blue staining (0.4%, Invitrogen) toassay cell viability of BHK21-15 cells (A) and C6/36 cells (B) after a45 min exposure to filtered Csp_P fresh biofilm. Difference in cellviability due to Csp_P exposure were non-significant for both cell lines(Mann Whitney Test).

FIG. 12. Exposure to Csp_P biofilm does not alter the insect cells'susceptibility to dengue virus. We filtered Csp_P biofilm using a 0.2-μmfilter and exposed C6/36 cells (grown to 80% confluency) to thebacterial filtrate for 45 min. Csp_P biofilm filtrate was then washedfrom the cells using 1×PBS, and cells were infected with dengue virus.Cells were assessed for plaque formation at 6 days post-infection.

FIG. 13. Csp_P biofilm is hemolytic when exposed to human red bloodcells. We mixed filtered Csp_P fresh biofilm with human erythrocytes,incubated 24 h at 37° C. and centrifuged at 2000 rpm for 5 min. We thenremoved the supernatant and assayed absorbance at 405 nm in an ELISAplate reader (HTS 7000 Perkin Elmer). 1×PBS was used as a negativecontrol and saponin as a positive control.

STATEMENT OF DEPOSIT

A biologically pure culture of Chromobacterium sp_Panamam (Csp_P) wasdeposited Sep. 4, 2014, under terms of the Budapest Treaty with theAmerican Type Culture Collection (ATCC®), 10801 University Blvd.,Manassas, Va. 20110, and given the accession number PTA-121570. For thepurposes of this invention, any isolate having the identifyingcharacteristics of strain Csp_P, including subcultures and variantsthereof which have the identifying characteristics and activity asdescribed herein are included.

DETAILED DESCRIPTION OF THE INVENTION

It is understood that the present invention is not limited to theparticular methods and components, etc., described herein, as these mayvary. It is also to be understood that the terminology used herein isused for the purpose of describing particular embodiments only, and isnot intended to limit the scope of the present invention. It must benoted that as used herein and in the appended claims, the singular forms“a,” “an,” and “the” include the plural reference unless the contextclearly dictates otherwise. Thus, for example, a reference to a“protein” is a reference to one or more proteins, and includesequivalents thereof known to those skilled in the art and so forth.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Specific methods, devices, andmaterials are described, although any methods and materials similar orequivalent to those described herein can be used in the practice ortesting of the present invention.

All publications cited herein are hereby incorporated by referenceincluding all journal articles, books, manuals, published patentapplications, and issued patents. In addition, the meaning of certainterms and phrases employed in the specification, examples, and appendedclaims are provided. The definitions are not meant to be limiting innature and serve to provide a clearer understanding of certain aspectsof the present invention.

The Gram-negative bacteria Chromobacterium sp_Panamam (Csp_P) wasisolated from the midgut of field collected Ae. Aegypti mosquitoes inPanama. The genus Chromobacterium spp. represents soil- andwater-associated bacteria of tropical and subtropical regions, andmembers of this genus are known to produce a variety of bioactivecompounds and to form biofilms. The most extensively studied member,Chromobacterium violaceum, has been found to produce violacein, a violetpigment compound with potent antimicrobial, anti-parasitic, andtumoricidal activity. Csp_P can be cultured in Luria Bertani (LB) broth(at 27-37° C.) and on LB agar, on which it forms flat colonies with atan color that becomes darker with time and are opaque when exposed tolight. Csp_P does not produce violacein, but molecular characterizationof its 16s rRNA gene sequence (SEQ ID NO:1) and phylogenetic analysisshowed a 98% similarity to Chromobacterium haemolyticum andChromobacterium aquaticum, probably its two closest relatives.

Mosquito gut colonization ability: Csp_P display an exceptional abilityto rapidly colonize midguts, showing a prevalence of 80% in An. Gambiaeand 97% in Ae. Aegypti cage populations at 3 days after exposure.Average bacterial loads at this time point were approximately 10⁵ and10⁴ per midgut in Ae. Aegypti and An. Gambiae females, respectively.

Entomopathogenic Activity: Supplementation of 2- to 4-day-old mosquitolarvae with 50 μl of a 1.0 OD600 liquid culture of Csp_P results inalmost complete mortality of An. Gambiae and Ae. Aegypti larvae over a3- and 2-day period, respectively, when compared to the control larvaethat were exposed to the normal breeding water microbiota.

Exposing antibiotic-treated An. Gambiae and Ae. Aegypti mosquitoes to asugar source for 24 hours containing Csp_P at a final concentration of10⁸ and 10⁶ CFU/ml, respectively, lead to a decrease in the longevity ofboth species when compared to non-exposed control mosquitoes. Similarly,a lower survival of septic (i.e., not pre-treated with antibiotics) An.Gambiae and Ae. Aegypti occurs after feeding on a blood meal containingCsp_P at a final concentration of 10⁸ CFU/ml.

Without being limited by any particular theory or mechanism, thesestudies suggest that Csp_P mediated mortality may be the result of amosquitocidal factor or systemic infection through dissemination intothe hemolymph; alternatively, its colonization of the midgut (or othertissues) might in some other way interfere with vital functions of themosquito.

In vivo (in mosquito) anti-dengue and anti-Plasmodium activity: An.Gambiae and Ae. Aegypti mosquitoes colonized with Csp_P through sugarfeeding prior to feeding on infectious blood displayed a significantlyincreased resistance to P. falciparum infection and dengue virusinfection. The inhibition of P. falciparum infection was even greaterwhen Csp_P was introduced through a blood meal at 10⁶ CFU/ml.

Csp_P exerts a direct anti-Plasmodium and anti-dengue effect in vitrothat is independent of the mosquito: Exposure of P. falciparumgametocytes to 42-h fresh biofilm filtrate results in 100% inhibition(p<0.001) and exposure to 42-h desiccated biofilm resulted inapproximately 60% inhibition (p<0.05, FIG. 5C) of gametocytedevelopment. Exposure of Plasmodium ookinete culture to Csp_P 48-hbiofilm (fresh and desiccated) and biofilm supernatant strongly blockedookinete development.

Exposure of dengue virus to Csp_P biofilm, desiccated biofilm or biofilmsupernatant abolishes dengue virus infectivity. Csp_P biofilm displaysstrong anti-dengue activity when the virus is suspended in human bloodand is dependent on biofilm maturation, since biofilm grown for 24 hoursshowed weaker inhibition when compared to 48 hour biofilm. The Csp_Pbiofilm-associated anti-Plasmodium and antiviral activity isheat-sensitive, since it can be inactivated through a 24-h incubation at90° C.

The anti-dengue activity of Csp_P biofilm is not a result of virusparticle sequestration by the biofilm, or a biofilm-mediated change inthe pH of the medium. Csp_P biofilm does not influence the host cells'susceptibility to dengue virus nor does it exert cytotoxic effects onhost cells. Chromobacterium sp_Panamam (Csp_P) is the first identifiedbacterium that exerts broad-spectrum anti-pathogen activity againstPlasmodium and dengue virus in their respective vectors, along withentomopathogenic activity against larval and adult stages of An. Gambiaeand Ae. Aegypti. Csp_P is the first bacterium that has been shown tomediate these diverse activities through secreted molecules. Thus, inparticular embodiments, the present invention provides mosquito controlproducts that target larval and adult stages of Anopheles and Aedesmosquitoes, though either direct exposure to the live or attenuatedbacterium, or to mosquitocidal extracts from this bacterium.

I. Definitions

The term “about” or “approximately” means within an acceptable errorrange for the particular value as determined by one of ordinary skill inthe art, which will depend in part on how the value is measured ordetermined, i.e., the limitations of the measurement system, i.e., thedegree of precision required for a particular purpose, such as apharmaceutical formulation. For example, “about” can mean within 1 ormore than 1 standard deviations, per the practice in the art.Alternatively, “about” can mean a range of up to 20%, up to 10%, up to5%, or up to 1% of a given value. Alternatively, particularly withrespect to biological systems or processes, the term can mean within anorder of magnitude, within 5-fold, within 4-fold, within 3-fold, orwithin 2-fold, of a value. Where particular values are described in theapplication and claims, unless otherwise stated the term “about” meaningwithin an acceptable error range for the particular value should beassumed.

The term “substantially,” as used herein, means at least about 80%, atleast about 85%, at least about 90%, at least about 91%, at least about92%, at least about 93%, at least about 94%, at least about 95%, atleast about 96%, at least about 97%, at least about 98%, at least about,or at least about 99%, including, for example, at least about 99.9%. Insome embodiments, the term “substantially” can mean completely, or about100%.

As used herein, the term “administering” encompasses any method by whichan insect can come into contact with a composition comprising Csp_P. Aninsect can be exposed to a composition by direct uptake (e.g., byfeeding). Alternatively, an insect can come into direct contact with acomposition comprising Csp_P. For example, an insect can come intocontact with a surface or material treated with a composition comprisingCsp_P. In certain embodiments, the terms can be used interchangeablywith the term “treating” or “treatment.”

As used herein the term “additional agent” refers to a small molecule,chemical, organic, or inorganic molecule that can be administered to orotherwise used to treat insects. In one embodiment, the “additionalagent” is a pesticide. As used herein, the term “pesticide” refers toany substance or mixture of substances intended for preventing,destroying, repelling, or mitigating any pest. A pesticide can be achemical substance or biological agent used against pests includinginsects that compete with humans for food, destroy property, spreaddisease, or are a nuisance. The term “additional agent” furtherencompasses other bioactive molecules such as antivirals pesticides,antifungals, antihelminthics, nutrients, sucrose and/or agents that stunor slow insect movement.

The term “whole broth culture” refers to a liquid culture containingboth cells and media. If bacteria are grown on a plate, the cells can beharvested in water or other liquid, whole culture.

The term “supernatant” refers to the liquid remaining when cells grownin broth or are harvested in another liquid from an agar plate and areremoved by centrifugation, filtration, sedimentation, or other meanswell known in the art.

The term “filtrate” refers to liquid from a whole culture that haspassed through a membrane.

The term “extract” refers to liquid substance removed from cells by asolvent (water, detergent, and buffer) and separated from the cells bycentrifugation, filtration or other method.

The term “metabolite” refers to a compound, substance or byproduct of afermentation of a microorganism, or supernatant, filtrate, or extractobtained from a microorganism that has insecticidal activity.

The term “insecticidal activity” means that a substance has adetrimental effect on an insect, including but not limited to killing atarget insect, increasing mortality, or inhibiting the incidence,growth, development or reproduction of a target insect.

II. Chromobacterium sp_Panamam (Csp_P)

The present inventors have discovered a new species of Chromobacteriumbacterium, which exhibits insecticidal activity against Anopheles andAedes mosquitoes. Cultures of the new bacterium are useful for controlof these and other insects. The species is designated as Chromobacteriumsp_Panamam (Csp_P).

The unique strain of the invention mediates insecticidal activity uponexposure to either larval or adult mosquito stages through the breedingwater or nectar meal, respectively. Without being limited by anyparticular theory or mechanism, these studies suggest that Csp_Pmediated mortality may be the result of a mosquitocidal factor orsystemic infection through dissemination into the hemolymph;alternatively, its colonization of the midgut (or other tissues) mightin some other way interfere with vital functions of the mosquito.

The full length Csp_P 16S rDNA gene sequence has been obtained and isshown in SEQ ID NO:1. The invention is also directed to Chromobacteriumstrains which have a 16S rDNA gene sequence of SEQ ID NO:1. Such strainsmay be isolated for example using appropriate nucleotide primers andidentified using the full length 16S rDNA gene sequence (SEQ ID NO:1).

The present invention is further directed to methods of controllinginsects using the unique bacterium of the invention. This aspectincludes application of an effective insect control amount of the straincells, supernatant, filtrate or extract containing an insecticidallyactive metabolite produced by the strain or combinations thereof. Csp_Phas been shown to reduce the survival of both the larval and adultAnopheles and Aedes mosquito stages.

A further aspect of the invention pertains to compositions whichincorporate the strain of the invention and/or compositions comprisingan insecticidally active metabolite produced by the strain of theinvention. Such compositions include, for example, whole cultures orsuspensions of the strain; supernatants, filtrates or extracts obtainedfrom the strain or combinations of the foregoing. Suchinsecticidally-active compositions may optionally include otheringredients such as an insect feeding stimulant, insect pheromone,insect attractant, fungicide, insecticide, photoactive dye, fluorescentbrighteners, spreading agent, sticking agent, thickener, emulsifier,stabilizer, preservative, buffer, water, diluent or other additive asknown in the art of formulation of insecticidal compositions.

The present invention is also directed to extracts obtained from thestrain which have insecticidal activity. Extraction from the cells isaccomplished using procedures known in the art. Exemplary proceduresinclude, but are not limited to, adding 0.1% detergent or 0.1% CHAPSbuffer to a cell pellet in equal volume of the original culture;extraction is for 30 minutes with shaking at room temperature. Cells areremoved by centrifugation; the supernatant contains the toxin. Theentire extract without removal of the cells is also toxic. Triton X-100can be used as the detergent in order to carry out tests for toxicity;however, other detergents can be used to extract the toxin. In aparticular embodiment, a volume of detergent or buffer to a cell pelletequal in volume to the original culture can be used for comparison oftoxicity; however, one could extract in a smaller volume and mayconcentrate the activity.

The present invention is further directed to methods of controllinginsects using the unique bacterium of the invention. This aspectincludes application of an effective insect control amount of thestrain, application of an effective insect control amount of asupernatant, filtrate or extract containing an insecticidally activemetabolite produced by the strain or application of combinations of theforegoing. The strain, supernatant, filtrate or extract is applied,alone or in combination, in an effective insect control or insecticidalamount. For the purposes of this invention, an effective amount isdefined as that quantity of microorganism cells, supernatant, filtrateor extract, alone or in combination, that is sufficient to kill thetarget insect, increase mortality, or inhibit the incidence, growth,development or reproduction of the target insect. Typically, aconcentration range about 1×10⁷ to about 1×10¹⁰ colony forming units(CFU)/ml is effective including about 1×10⁷, 2×10⁷, 3×10⁷ 4×10⁷, 5×10⁷,6×10⁷, 7×10⁷, 8×10⁷, 9×10⁷, 1×10⁸, 2×10⁸, 3×10⁸, 4×10⁸, 5×10⁸, 6×10⁸,7×10⁸, 8×10⁸, 9×10⁸, 1×10⁹, 2×10⁹, 3×10⁹, 4×10⁹, 5×10⁹, 6×10⁹, 7×10⁹,8×10⁹, 9×10⁹, 1×10¹⁰, 2×10¹⁰, 3×10¹⁰, 4×10¹⁰, 5×10¹⁰, 6×10¹⁰, 7×10¹⁰,8×10¹⁰, 9×10¹⁰, or 1×10¹¹ or more CFU/ml is effective. The effectiverate can be affected by insect species present, stage of insect growth,insect population density, and environmental factors such astemperature, wind velocity, rain, time of day and seasonality. Theamount that will be within an effective range in a particular instancecan be determined by laboratory or field tests.

III. Administration of Compositions Comprising Csp_P to Insects

An insect (e.g., an Anopheles or Aedes mosquito) can be exposed to acomposition comprising Csp_P in combination with a delivery agent in anysuitable manner that permits administering the composition to theinsect. For example, the insect can be contacted with the composition ina pure or substantially pure form, for example a solution containingCsp_P. In a particular embodiment, the composition comprises Csp_P and adelivery agent. In another particular embodiment, the insect can besimply “soaked” or “sprayed” with a solution comprising Csp_P.

Alternatively, the composition comprising Csp_P can be linked to a foodcomponent of the insect, such as artificial nectar or sugar bait, forease of delivery and/or in order to increase uptake of the compositionby the insect. Methods for oral introduction include, for example,directly mixing a composition with the insect's food, spraying thecomposition in the insect's habitat or field including standing waterareas. The composition can also be incorporated into the medium in whichthe insect grows, lives, reproduces, feeds, or infests.

In another embodiment, the composition is in the form of a bait. Thebait is designed to lure the insect to come into contact with thecomposition. In one embodiment, upon coming into contact therewith, thecomposition is then internalized by the insect, by ingestion forexample. The bait can depend on the species being targeted. Anattractant can also be used. The attractant can be a pheromone, such asa male or female pheromone. The attractant acts to lure the insect tothe bait, and can be targeted for a particular insect or can attract awhole range of insects. The bait can be in any suitable form, such as asolid, paste, pellet or powdered form.

The bait can also be carried away by the insect back to the colony. Thebait can then act as a food source for other members of the colony, thusproviding an effective control of a large number of insects andpotentially an entire insect pest colony.

The baits can be provided in a suitable “housing” or “trap”. Suchhousings and traps are commercially available and existing traps can beadapted to include the compositions of the invention. The housing ortrap can be box-shaped for example, and can be provided in pre-formedcondition or can be formed of foldable cardboard for example. Suitablematerials for a housing or trap include plastics and cardboard,particularly corrugated cardboard. The inside surfaces of the traps canbe lined with a sticky substance in order to restrict movement of theinsect once inside the trap. The housing or trap can contain a suitabletrough inside which can hold the bait in place. A trap is distinguishedfrom a housing because the insect cannot readily leave a trap followingentry, whereas a housing acts as a “feeding station” which provides theinsect with a preferred environment in which they can feed and feel safefrom predators.

In certain embodiments of the invention, an area can be treated with acomposition of the present invention, for example, by using a sprayformulation, such as an aerosol or a pump spray. In certain embodimentsof the invention, an area can be treated, for example, via aerialdelivery, by truck-mounted equipment, or the like. Of course, varioustreatment methods can be used without departing from the spirit andscope of the present invention. In some embodiments, the composition issprayed by e.g., backpack spraying, aerial spraying, spraying/dustingetc.

In specific embodiment, treatment can include use of an oil-basedformulation, a water-based formulation, a residual formulation, and thelike. In some embodiments, combinations of formulations can be employedto achieve the benefits of different formulation types.

In further embodiments, the compositions and methods of the presentinvention can be used to control other insects. As used herein the term“insect” describes any insect, meaning any organism belonging to theKingdom Animals, more specific to the Phylum Arthropoda, and to theClass Insecta or the Class Arachnida. In specific embodiments of thepresent invention, the insect can belong to the following orders: Acari,Araneae, Anoplura, Coleoptera, Collembola, Dermaptera, Dictyoptera,Diplura, Diptera, Embioptera, Ephemeroptera, Grylloblatodea, Hemiptera,Homoptera, Hymenoptera, Isoptera, Lepidoptera, Mallophaga, Mecoptera,Neuroptera, Odonata, Orthoptera, Phasmida, Plecoptera, Protura,Psocoptera, Siphonaptera, Siphunculata, Thysanura, Strepsiptera,Thysanoptera, Trichoptera, and Zoraptera.

As used herein, the terms “pest” or “insect pests” include but are notlimited to the following examples: from the order Lepidoptera, forexample, Acleris spp., Adoxophyes spp., Aegeria spp., Agrotis spp.,Alabama argillaceae, Amylois spp., Anticarsia gemmatalis, Archips spp,Argyrotaenia spp., Autographa spp., Busseola fusca, Cadra cautella,Carposina nipponensis, Chilo spp., Choristoneura spp., Clysiaambiguella, Cnaphalocrocis spp., Cnephasia spp., Cochylis spp.,Coleophora spp., Crocidolomia binotalis, Cryptophlebia leucotreta, Cydiaspp., Diatraea spp., Diparopsis castanea, Earias spp., Ephestia spp.,Eucosma spp., Eupoecilia ambiguella, Euproctis spp., Euxoa spp.,Grapholita spp., Hedya nubiferana, Heliothis spp., Hellula undalis,Hyphantria cunea, Keiferia lycopersicella, Leucoptera scitella,Lithocollethis spp., Lobesia botrana, Lymantria spp., Lyonetia spp.,Malacosoma spp., Mamestra brassicae, Manduca sexta, Operophtera spp.,Ostrinia Nubilalis, Pammene spp., Pandemis spp., Panolis flammea,Pectinophora gossypiella, Phthorimaea operculella, Pieris rapae, Pierisspp., Plutella xylostella, Prays spp., Scirpophaga spp., Sesamia spp.,Sparganothis spp., Spodoptera spp., Synanthedon spp., Thaumetopoea spp.,Tortrix spp., Trichoplusia ni and Yponomeuta spp.; from the orderColeoptera, for example, Agriotes spp., Anthonomus spp., Atomarialinearis, Chaetocnema tibialis, Cosmopolites spp., Curculio spp.,Dermestes spp., Epilachna spp., Eremnus spp., Leptinotarsa decemlineata,Lissorhoptrus spp., Melolontha spp., Orycaephilus spp., Otiorhynchusspp., Phlyctinus spp., Popillia spp., Psylliodes spp., Rhizopertha spp.,Scarabeidae, Sitophilus spp., Sitotroga spp., Tenebrio spp., Triboliumspp. and Trogoderma spp.; from the order Orthoptera, for example, Blattaspp., Blattella spp., Gryllotalpa spp., Leucophaea maderae, Locustaspp., Periplaneta ssp., and Schistocerca spp.; from the order Isoptera,for spp; from the order Psocoptera, for spp.; from the order Anoplura,for example jfaematopinus spp., Linognathus spp., Pediculus spp.,Pemphigus spp. and Phylloxera spp.; from the order Mallophaga, forexample Trichodectes spp.; from the order Thysanoptera, for spp.,Hercinothrips spp., Taeniothrips spp., Thrips palmi, Thrips tabaci andScirtothrips aurantii; from the order Heteroptera, for example, Cimexspp., Distantiella theobroma, Dysdercus spp., Euchistus spp., Eurygasterspp., Leptocorisa spp., Nezara spp., Piesma spp., Rhodnius spp.,Sahlbergella singularis, Scotinophara spp., Triatoma spp., Miridaefamily spp. such as Lygus hesperus and Lygus lineoloris, LygaeidaQfamily spp. such as Blissus leucopterus, and Pentatomidae family spp.;from the order Homoptera, for example, Aleurothrixus floccosus,Aleyrodes brassicae, Aonidiella spp., Aphididae, Aphis spp., Aspidiotusspp., Bemisia tabaci, Ceroplaster spp., Chrysomphalus aonidium,Chrysomphalus dictyospermi, Coccus hesperidum, Empoasca spp., Eriosomalarigerum, Erythroneura spp., Gascardia spp., Laodelphax spp., Lacaniumcorn, Lepidosaphes spp., Macrosiphus spp., Myzus spp., Nehotettix spp.,Nilaparvata spp., Paratoria spp., Pemphigus spp., Planococcus spp.,Pseudaulacaspis spp., Pseudococcus spp., Psylla ssp., Pulvinariaaethiopica, Quadraspidiotus spp., Rhopalosiphum spp., Saissetia spp.,Scaphoideus spp., Schizaphis spp., Sitobion spp., Trialeurodesvaporariorum, Trioza erytreae and Unaspis citri; from the orderHymenoptera, for example, Acromyrmex, Atta spp., Cephus spp., Diprionspp., Diprionidae, Gilpinia polytoma, Hoplocampa spp., Lasius spp.,Monomorium pharaonis, Neodiprion spp, Solenopsis spp. and Vespa ssp.;from the order Diptera, for example, Aedes spp., Anopheles spp.,Antherigona soccata, Bibio hortulanus, CalHphora erythrocephala,Ceratitis spp., Chrysomyia spp., Culex spp., Cuterebra spp., Dacus spp.,Drosophila melanogaster, Fannia spp., Gastrophilus spp., Glossina spp.,Hypoderma spp., Hyppobosca spp., Liriomysa spp., Lucilia spp.,Melanagromyza spp., Musca ssp., Oestrus spp., Orseolia spp., Oscinellafrit, Pegomyia hyoscyami, Phorbia spp., Rhagoletis pomonella, Sciaraspp., Stomoxys spp., Tabanus spp., Tannia spp. and Tipula spp., from theorder Siphonaptera, for example, Ceratophyllus spp. and Xenopsyllacheopis and from the order Thysanura, for example Lepisma saccharin.

In other embodiments, a composition comprising Csp_P can be administeredto an insect including, but not limited to, those with piercing-suckingmouthparts, as found in Hemiptera and some Hymenoptera and Diptera suchas mosquitoes, bees, wasps, lice, fleas and ants, as well as members ofthe Arachnidae such as ticks and mites; order, class or family ofAcarina (ticks and mites) e.g., representatives of the familiesArgasidae, Dermanyssidae, Ixodidae, Psoroptidae or Sarcoptidae andrepresentatives of the species Amblyomma spp., Anocentor spp., Argasspp., Boophilus spp., Cheyletiella spp., Chorioptes spp., Demodex spp.,Dermacentor spp., Dermanyssus spp., Haemophysalis spp., Hyalomma spp.,Ixodes spp., Lynxacarus spp., Mesostigmata spp., Notoedres spp.,Ornithodoros spp., Ornithonyssus spp., Otobius spp., otodectes spp.,Pneumonyssus spp., Psoroptes spp., Rhipicephalus spp., Sarcoptes spp.,or Trombicula spp.; Anoplura (sucking and biting lice) e.g.,representatives of the species Bovicola spp., Haematopinus spp.,Linognathus spp., Menopon spp., Pediculus spp., Pemphigus spp.,Phylloxera spp., or Solenopotes spp.; Diptera (flies) e.g.,representatives of the species Aedes spp., Anopheles spp., Calliphoraspp., Chrysomyia spp., Chrysops spp., Cochliomyia spp., Cw/ex spp.,CuUcoides spp., Cuterebra spp., Dermatobia spp., Gastrophilus spp.,Glossina spp., Haematobia spp., Haematopota spp., Hippobosca spp.,Hypoderma spp., Lucilia spp., Lyperosia spp., Melophagus spp., Oestrusspp., Phaenicia spp., Phlebotomus spp., Phormia spp., Sarcophaga spp.,Simulium spp., Stomoxys spp., Tabanus spp., Tannia spp. or Zzpu/alphaspp.; Mallophaga (biting lice) e.g., representatives of the speciesDamalina spp., Felicola spp., Heterodoxus spp. or Trichodectes spp.; orSiphonaptera (wingless insects) e.g., representatives of the speciesCeratophyllus spp., Xenopsylla spp; Cimicidae (true bugs) e.g.,representatives of the species Cimex spp., Tritominae spp., Rhodiniusspp., or Triatoma spp.

Embodiments of the present invention can be used to control parasites.As used herein, the term “parasite” includes parasites, such as but notlimited to, protozoa, including intestinal protozoa, tissue protozoa,and blood protozoa. Examples of intestinal protozoa include, but are notlimited to: Entamoeba hystolytica, Giardia lamblia, Cryptosporidiummuris, and Cryptosporidium parvum. Examples of tissue protozoa include,but are not limited to: Trypanosomatida gambiense, Trypanosomatidarhodesiense, Trypanosomatida crusi, Leishmania mexicana, Leishmaniabraziliensis, Leishmania tropica, Leishmania donovani, Toxoplasmagondii, and Trichomonas vaginalis. Examples of blood protozoa include,but are not limited to Plasmodium vivax, Plasmodium ovale, Plasmodiummalariae, and Plasmodium falciparum. Histomonas meleagridis is yetanother example of a protozoan parasite.

Without further elaboration, it is believed that one skilled in the art,using the preceding description, can utilize the present invention tothe fullest extent. The following examples are illustrative only, andnot limiting of the remainder of the disclosure in any way whatsoever.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how thecompounds, compositions, articles, devices, and/or methods described andclaimed herein are made and evaluated, and are intended to be purelyillustrative and are not intended to limit the scope of what theinventors regard as their invention. Efforts have been made to ensureaccuracy with respect to numbers (e.g., amounts, temperature, etc.) butsome errors and deviations should be accounted for herein. Unlessindicated otherwise, parts are parts by weight, temperature is indegrees Celsius or is at ambient temperature, and pressure is at or nearatmospheric. There are numerous variations and combinations of reactionconditions, e.g., component concentrations, desired solvents, solventmixtures, temperatures, pressures and other reaction ranges andconditions that can be used to optimize the product purity and yieldobtained from the described process. Only reasonable and routineexperimentation will be required to optimize such process conditions.

Example 1: Chromobacterium Csp_P Reduces Malaria and Dengue Infection inVector Mosquitoes

The influence of the gut microbiota on the vector competence of diseasevectors such as mosquitoes has gained increasing interest over the pastdecade. Previous work has shown that co-infection of Anophelesmosquitoes with Plasmodium and with Serratia sp. or Enterobacter sp.bacteria leads to reduced Plasmodium infection. Additionally, thepresence of certain bacterial species in Aedes mosquito midguts leads toa lower intensity of dengue virus infection. Studies have also shownthat Anopheles and Aedes mosquitoes that have had their gut microbiotaexperimentally reduced via antibiotic treatment show higher Plasmodiumand dengue virus infection levels, respectively, than do their untreatedcounterparts. The anti-pathogen activity of mosquito midgut bacteria hasbeen attributed to the elicitation of the mosquito immune system in someinstances, and to direct anti pathogenic activity of bacteria-producedmolecules in others. Activation of the immune deficiency (IMD) pathway,the major anti-P. falciparum immune pathway, has been shown to bemediated through an interaction between the pattern recognition receptorPGRP-LC and the midgut microbiota. In turn, microbe-derivedanti-pathogen factors have been characterized in some microbe-hostinteraction systems and include cytotoxic metalloproteases, hemolysins,antibiotics, haemaglutinins, proteases, prodigiosin pigments, and ironchelators (siderophores).

In nature, bacteria commonly grow attached to surfaces in complexmatrices of cells, proteins, polysaccharides, and DNA (biofilm growth),rather than as single free-swimming cells (planktonic growth). Biofilmformation allows the bacteria to survive exposure to host-derivedantimicrobial factors and other environmental stressors. Furthermore,bacterial cells in a biofilm have quite different gene expression andmetabolic profiles than do cells in a free-swimming planktonic state.Studies of Pseudomonas aeruginosa colonization of the Drosophilamelanogaster gut have shown that biofilm formation can dramaticallyaffect dissemination in the hemolymph and fly mortality.

In this study, we show that a Chromobacterium sp_Panamam isolate, Csp_P,isolated from the midgut of field-collected Ae. aegypti mosquitoes,exerts in vitro anti-Plasmodium and anti-dengue activity when grownunder biofilm conditions. Csp_P can effectively colonize the intestinesof the two most important mosquito disease vectors, An. gambiae and Ae.aegypti, where it blocks Plasmodium and dengue infection. It also exertsentomopathogenic activity against both larval and adult stages and couldtherefore be used for the development of a biocontrol agent. Csp_P'santi-pathogen activities appear to be mediated by stable secondarymetabolites, suggesting that Csp_P is a source of potentiallyinteresting candidates for the development of therapeutic andtransmission-blocking drugs.

Materials and Methods

Ethics Statement.

This study was carried out in strict accordance with the recommendationsin the Guide for the Care and Use of Laboratory Animals of the NationalInstitutes of Health. Mice were only used for mosquito rearing as ablood source according to approved protocol. The protocol was approvedby the Animal Care and Use Committee of the Johns Hopkins University(Permit Number: M006H300). Commercial anonymous human blood, suppliedfrom Interstate Blood Bank Inc., was used for Plasmodium and denguevirus infection assays in mosquitoes, and informed consent was thereforenot applicable. The Johns Hopkins School of Public Health EthicsCommittee has approved this protocol. Mosquito collections wereperformed in residences after owners/residents permission.

Mosquito Rearing and Antibiotic Treatment.

Aedes aegypti mosquitoes were from the Rockefeller strain, and Anophelesgambiae mosquitoes were from the Keele strain. Both were maintained on a10% sugar solution at 27° C. and 95% humidity with a 12-h light/darkcycle. Sterile cotton, filter paper, and sterilized nets were used tomaintain the cages as sterilely as possible. For experiments utilizingaseptic mosquitoes, females were maintained on a 10% sucrose solutionwith 20 U penicillin and 20 μg streptomycin from the first daypost-eclosion until 1-2 days prior to challenge. The effectiveness ofthe antimicrobial treatment was confirmed by colony forming unit assaysprior to blood-feeding or bacterial challenge.

Introduction of Bacteria Via Sugar Meal.

In cases where mosquitoes were antibiotic treated, reintroduction ofbacteria through a sugar meal was done by first treating mosquitoes withantibiotics for 2-3 days after emergence, then providing them with 10%sucrose (for An. gambiae) or sterile water (for Ae. aegypti) for 24 hpost-antibiotic treatment. When mosquitoes were not antibiotic treated,they were maintained on 10% sucrose for 2-5 days post emergence. Ae.aegypti were given sterile water during the final 24 hours of thisperiod. In all cases, mosquitoes were then starved overnight and fed for24 h on cotton strips moistened with a 1.5% sucrose solution containingCsp_P at a final concentration of approximately 10⁸ CFU/ml for An.gambiae and 10⁶ CFU/ml for Ae. aegypti. In some experiments (FIGS. 1 and2), Ae. aegypti mosquitoes were also fed Csp_P at a final concentrationof 10¹⁰ CFU/ml.

Assaying Prevalence and Bacterial Load of Csp_P.

In antibiotic treated mosquitoes, midguts were dissected three days postingestion of Csp_P, homogenized in 1×PBS and plated on LB agar. Colonieswere then counted to estimate colony forming units (CFUs) per midgut aswell as prevalence of Csp_P. In mosquitoes not treated with antibiotics,prevalence and/or bacterial load was estimated in one of two ways. ForAn. gambiae, midguts were dissected at one and two days post Csp_Pingestion, homogenized in 1×PBS and serial dilutions of the homogenatewere plated on LB agar supplemented with ampicillin (10,000 ug/ml).Csp_P is highly resistant to ampicillin and grows readily even at thishigh concentration. We verified that Csp_P was the only bacteriumgrowing on antibiotic treated plates by first confirming that allcolonies that grew were similar in color, growth rate and colonymorphology. 16s rDNA was then sequenced from a subset of colonies andverified to match the sequence of Csp_P from pure freezer stock.

It was not possible to use this method for Ae. aegypti because theirmidguts commonly contained other highly ampicillin-resistant bacteria.These contaminants grew to very high numbers on the ampicillin-treatedplates and interfered with the detection of Csp_P. DNA was thereforeextracted using the ZR Soil Microbe DNA MicroPrep kit (Zymo Research)from samples dissected 1 and 3 days after feeding on a sugar mealcontaining either PBS or Csp_P (10¹⁰ CFU/ml). The manufacturer'sprotocol was altered in the following way: instead of using lysis bufferto disrupt cells, each midgut was put in 500 μl 1×PBS, 25 μl lysozyme(10 mg/ml) and 7.5 μl mutanolysin (10 KU/ml) were added and the sampleswere incubated at 37° C. for 1.5 h. 15 μl proteinase K and 25 μl 10% SDSwere then added, samples were incubated at 55° C. for 1 h, and thestandard protocol was then resumed. A diagnostic PCR was performed toassess the presence of Csp_P in each individual midgut. Primers weredesigned to amplify a 415 bp fragment of the Csp_P hydrogen cyanidesynthase B gene and the primers were verified to be Csp_P-specific usingPrimer BLAST from NCBI (Forward primer: 5′AGGGCGTAACCCTGGACTAT 3′ (SEQID NO:2), Reverse primer: 5′ CCGAAGGAACTGGCTTCGTA 3′ (SEQ ID NO:3)). PCRwas performed with the above primers using 10 ng DNA as template andPhusion HighFidelity DNA Polymerase according to the manufacturer'sinstructions, with the following exceptions: 0.5 μl of each primer (10μm) was used, and 0.25 μl BSA was added to each reaction. Cyclingconditions were as follows: 95° C. for 30 seconds, [95° C. for 30 s, 65°C. for 30 s, 72° C. for 45 s]×27 cycles, 72° C. for 10 minutes. 8 μl ofeach sample was run on a 1% agarose gel and visualized at 400 msexposure. A visible 415 bp band was considered positive evidence ofCsp_P bacteria (data not shown). A very faint band was detected in oneof 40 PBS samples, suggesting a minor contamination event or thepresence of another bacterium with high sequence identity to Csp_P. Thiswas an isolated incident and was not seen in any other PBS samples. Twoindependent PCR products were sequenced from Csp_P fed samples andverified to be a perfect match to the sequence obtained from Csp_Psequenced directly from freezer stock. To serve as a positive controland to allow estimation of the sensitivity of the diagnostic PCR, astandard curve was run in which a range of 10⁷-10¹ copies of the Csp_Phcn B PCR product was used as template. In this way, it was possible toestimate the minimum detection threshold of this assay. Using the abovementioned PCR conditions, a band was detectable in wells containing 10³initial copies of the hcn B product but not in wells containing 10²initial copies, suggesting that this assay is capable of detecting aminimum of 10³ copies of Csp_P/midgut.

Introduction of Bacteria Via Blood Meal.

At 2 days prior to blood feeding, sucrose was removed, and themosquitoes were given sterile water. They were then starved for 12 hprior to blood feeding. Csp_P was grown overnight in liquid LB at 30° C.The overnight culture (1 ml) was then pelleted, washed with 1×PBS, andresuspended in 1×PBS to OD600=1.0, which equals a concentration ofapproximately 10⁸ CFU/ml. Mosquitoes were then allowed to membrane-feedon blood containing bacteria or 1×PBS as a control (blood mixture: 50%1.0 OD600 bacterial culture or 1×PBS, 40% blood, 10% human serum).Bacteria-fed adult females ingested approximately 10⁵ CFU per mosquito.

Exposure of Larvae to Csp_P.

At 2-4 days post-hatching, larvae were placed in cell culture plates ingroups of 10 per well. Each well contained 5 ml sterile water plus asmall amount of larval food (liver powder, tropical fish flake food, andrabbit food pellets mixed in a 2:1:1 ratio). We then added 50 μl of anovernight culture of Csp_P diluted to OD600=1.0 (10⁸ CFU/ml) to eachwell; 1×PBS was added to control wells, and mortality was monitored inall wells for a 5 day period.

Cell Culture Maintenance, Mosquito Infections with Dengue Virus, andTitration of Infected Midgets.

Dengue virus serotype 2 (New Guinea C strain, DENV-2) was propagated inthe C6/36 mosquito cell line according to previously published methods.In brief, cell line infection was allowed to proceed for 5-7 days, atwhich time the cells were harvested with a cell scraper and lysed byfreezing and thawing in dry CO2 and a 37° C. water bath, thencentrifuged at 800 g for 10 min. Dengue virus serotype 2 was isolatedand mixed 1:1 with commercial human blood and used for infections asdescribed in. Mosquitoes that had previously fed on Csp_Pbacteria-sucrose solution were starved overnight prior to dengue virusinfection. Infected mosquitoes were collected at 7 days post-infectionand surface-sterilized by dipping them in 70% ethanol for 1 min and thenrinsing them twice in 1×PBS for 2 min each. Midgut dissection was donein one drop of 1×PBS under sterile conditions, and the midgut wastransferred to a microcentrifuge tube containing 150 μl of MEM. Midgutswere homogenized using a Kontes pellet pestle motor, filtered, andstored at −80° C. until ready for virus titration.

Dengue virus titration of infected midguts was done as previouslyreported. In brief, the infected midgut homogenates were seriallydiluted and inoculated into C6/36 cells in 24-well plates. After anincubation of 5 days at 32° C. and 5% CO2, the plates were fixed with50%/50% methanol/acetone, and plaques were assayed by peroxidaseimmunostaining using mouse hyperimmune ascitic fluid specific for DENV-2as the primary antibody and a goat anti-mouse HRP conjugate as thesecondary antibody. In addition, where indicated, dengue virus plaqueassays were conducted in BHK-21 cells. At 5 days post-infection, the24-well plates were fixed and stained with crystal violet. Plaques(formed by cells with cytopathic effect) were counted and analyzed.

P. falciparum Cultivation, Mosquito Infections, and Oocyst Counts.

P. falciparum strain NF54 was maintained in continuous culture accordingto the method described by Tragger and Jensen, 192 SCIENCE 673-75(1976). In brief, P. falciparum was grown in O+ red blood cells (RBCs)at 2% hematocrit and RPMI 1640 medium supplemented with glutamine,HEPES, hypoxanthine, and 10% O+ human serum. To maintain amicroaerophilic environment, parasites were maintained in a candle jarat 37° C. Use of human erythrocytes to support the growth of P.falciparum was approved by the internal review board of the BloombergSchool of Public Health. Gametocytemia and exflagellation events wereassessed after 18 days of P. falciparum culture. The gametocyte culturewas centrifuged and diluted in a mixture of RBCs supplemented withserum. Mosquitoes were rendered aseptic via antibiotic treatment andthen fed on membrane feeders for 30 min with blood containing P.falciparum gametocytes. Csp_P was either added directly to theinfectious blood meal (bacterial concentration=10⁶ CFU/mL) or introducedvia sugar meal as described above 3 to 4 days prior to the infectiousblood meal. On the same day as the blood meal, mosquitoes were sorted,and the unfed mosquitoes were removed. At 7 to 8 days after bloodfeeding, the fed mosquitoes were dissected, and their midguts werestained with 0.1% mercurochrome. The number of oocysts per midgut wasdetermined with a light contrast microscope, and the median wascalculated for the control and each experimental condition. More thanthree independent replicates were used per group.

Csp_P Culture Preparations for In Vitro Anti-Plasmodium and Anti-DengueActivity Assays.

To grow bacteria in planktonic conditions, we spiked 5 ml sterile LBwith 5 μl of bacterial freezer stock and allowed the culture to growovernight at 30° C. with shaking. We then diluted planktonic cultures toOD600=1.0 (±0.1) with additional sterile LB broth which, for Csp_P,results in a concentration of approximately 10⁸ CFU/ml. To grow bacteriaunder biofilm conditions, we dispensed 1 ml of sterile LB into each wellof a 24-well cell culture plate and spiked each well with 1 μl ofbacterial freezer stock. We then allowed the culture to grow at roomtemperature without shaking for 48 h. Csp_P biofilm supernatant washarvested from single bacterial culture wells containing 48-h biofilmand was found to have an average bacterial concentration ofapproximately 10⁹ CFU/ml. To harvest fresh biofilm, we removed thesupernatant from five wells containing 48-h biofilm, resuspended thebiofilm from each well in 100 μl 1×PBS and pooled the five wells. ForCsp_P, this pooled biofilm solution contained approximately 10⁹ CFU/mland an average of 5 mg of biofilm (dry weight). To obtain desiccatedbiofilm, we collected the fresh biofilm from five wells as indicated,centrifuged the biofilm at 5000 rpm for 2.5 min, removed the PBSsupernatant, and allowed the biofilm to dry at room temperature. On theday of the experiment, we resuspended the five wells of desiccatedbiofilm in 500 μl 1×PBS to mimic the fresh biofilm treatment. Toheat-inactivate the fresh biofilm, we collected fresh biofilm asindicated and incubated samples at 90° C. for 24 h prior to theexperiment.

In Vitro Anti-Plasmodium Activity Assays.

We prepared Csp_P bacterial cultures as described above and filtered allsamples through a 0.2-μm filter. Asexual-stage assay: Inhibition ofasexual-stage P. falciparum was assessed using a SYBR green I-basedfluorescence assay as described earlier. Csp_P biofilm was grown for 36h for this experiment because 48-h biofilm causes hemolysis of RBCs(FIG. 13), which interferes with the assay. Parasites were synchronizedusing 5% sorbitol; 5 μl of each bacterial preparation was dispensed intriplicate wells of 96-well microplates, followed by addition of 95 μlof synchronous ring-stage P. falciparum cultures at 1% hematocrit and 1%parasitemia. Chloroquine (250 nM) was used as a positive control, andparasite growth medium was used as a negative control. After 72 h ofincubation in a candle jar at 37° C., an equal volume of SYBR green-Isolution in lysis buffer (Tris [20 mM; pH 7.5], EDTA [5 mM], saponin[0.008%; w/v], and Triton X-100 [0.08%; v/v]) was added to each well andmixed gently, then incubated 1-2 h in the dark at room temperature.Plates were read on a fluorescence plate reader (HTS 7000, PerkinElmer), with excitation and emission wavelengths of 485 and 535 nm,respectively. Percent inhibition was calculated relative to negative (0%inhibition) and positive controls (100% inhibition). Three biologicalreplicates were assayed.

Ookinete-Stage Assay:

To assess inhibition of ookinete-stage P. berghei parasites, femaleSwiss Webster mice (6-8 weeks old) were infected with a transgenicstrain of P. berghei that expresses Renilla luciferase. Starting at 3days post-infection, exflagellation assays were performed until at least20 exflagellation events were recorded in a 20× field. At this time,mice were bled by heart puncture using a heparinized needle, and theblood was diluted in 10 volumes of ookinete medium (RPMI 1640, 10% FBS,50 mg/ml hypoxanthine, and 2 mg/ml NaHC0³, pH 8.3) with 4% mouse RBClysate. Samples (50 μl) of each bacterial preparation were then mixedwith the infected blood and incubated for 24 h at 19° C. Ookinete countswere determined using the Renilla luciferase assay system (Promega, USA)according to the manufacturer's instructions. The experiment wasperformed on two independent days, and each sample was assayed intriplicate on each day.

Gametocyte-Stage Assay:

Inhibition of gametocyte-stage P. falciparum by Csp_P was assessed asdescribed previously. To prevent hemolysis of RBCs, Csp_P biofilm wasgrown for 36 and 42 hours for this experiment. In brief, NF54 P.falciparum cultures were started at 0.5% asexual parasitemia and 4%hematocrit. Csp_P bacterial preparations were added 15 days afterPlasmodium cultures were initiated, and gametocytemia was determined 18days after culture initiation. At least three biological replicates weretested for each culture preparation. More than 500 erythrocytes wereexamined for gametocytes across Giemsa-stained blood films from eachsample.

In Vitro Anti-Dengue Activity Assays.

We prepared Csp_P bacterial cultures as described above (planktonicstate, biofilm, biofilm supernatant, desiccated biofilm, andheat-inactivated biofilm), mixed 75 μl of each bacterial culturepreparation with 75 μl of MEM containing dengue virus serotype 2 andincubated the mixture at room temperature for 45 min. Samples were thenfiltered through a 0.2-μm filter, serially diluted, and used to infectBHK21-15 cells. Plaque assays were conducted as described above toassess dengue virus infectivity. Percent inhibition was calculated asthe percent decrease in PFU/ml relative to the PBS+LB control, which wasstandardized to 0% inhibition. The experiment was performed on twoindependent days, and each assay was performed in triplicate on eachday. In experiments in which dengue was mixed with human blood beforeexposure to Csp_P, bacterial biofilms were not removed from the cellculture plate. Rather, dengue virus was mixed 1:1 with human blood, and150 μl of this mixture was added directly to each well containing Csp_Pbiofilm and incubated for 45 min at 30° C. Following this incubationperiod, the blood-dengue virus solution was mixed with the biofilm, and50 μl of the mixture was then drawn from the well, diluted in MEM, andfiltered through a 0.2-μm filter. The resulting filtrate solution wasthen serially diluted and used to infect C6/36 cells.

Assay for Sequestration of Viral Particles by Csp_P Biofilm.

To assess whether the antidengue activity of Csp_P was due tosequestration of DENV by the Csp_P biofilm, we mixed a dengue virussuspension with Csp_P 48 hr biofilm or LB broth and incubated it for aperiod of 45 min. Samples were then centrifuged at 5,000 rpm for 5 min.The supernatants were collected, and RNA was extracted from equalvolumes (50 μl) of experimental (biofilm+DENV) and control (LB+DENV)samples using the RNeasy kit (Qiagen). Comparison of viral RNA loads inthe extracted supernatant was done via RT-qPCR relative quantification,using 2 μl of the viral RNA in a 20-μl reaction volume.

Assessing pH Effects on Dengue Virus Infectivity.

The pH of bacterial biofilms and supernatants was assessed with amicro-pH electrode (Lazar Lab) at room temperature. Effects of pHchanges on dengue virus infectivity were assessed by adjusting the pH ofthe MEM with NaOH and HCl until the desired range of pH values wasobtained: 5.0, 7.7, 8.5, and 10.0. The pH-adjusted MEM was then mixedwith dengue virus-laden blood and incubated for 45 min prior to serialdilution and infection of C6/36 cells.

Cell Viability Assays.

Cell viability assays on the mosquito cell line C6/36 and the vertebratecell line BHK-21 were performed via trypan blue staining (0.4%,Invitrogen) according to the manufacturer's instructions. In brief, 50μl of suspended cells were placed in a microcentrifuge tube and mixedwith 10 μl of Csp_P filtered fresh biofilm or PBS as a control. C6/36cells were incubated at 32° C. and BHK-21 cells were incubated at 37°C.+5% CO2 for 45 min. Cells were then mixed with 12 μl of 0.4% trypanblue stain. The mixture was allowed to stand for 5 min at roomtemperature and then loaded into a hemocytometer for cell viabilityassessment and counting under a microscope.

Assay of the Effects of Csp_P Biofilm on Host Cell Susceptibility toDENV.

To assess whether exposure to Csp_P biofilm changes the susceptibilityof the host cell to DENV, we conducted assays exposing C6/36 cells toCsp_P-filtered biofilm prior to dengue virus infection. Cells were grownto 80% confluency; the cell medium was then removed, washed once with1×PBS, and then overlaid with 100 μl of Csp_P biofilm that had beenfiltered using a 2-μm filter or with 1×PBS (control) for about 10 min.Plates were then washed three times with 1×PBS and then infected with100 μl of dengue virus for about 45 min. Cells were assessed for plaqueformation at 6 days post-infection.

Hemolysis Assay.

Human erythrocytes were washed with RPMI 1640 medium until thesupernatant was visually free of hemoglobin pigment. The washederythrocytes were suspended in malaria complete medium to yield a 1%hematocrit. Filtered Csp_P biofilm was mixed with erythrocytes andincubated up to 24 h at 37° C. To separate lysed RBC cytosol from wholeRBCs, the suspension was centrifuged at 2000 rpm for 5 min. Theresulting supernatant was carefully aspirated and plated in new 96-wellmicroplates. Control erythrocytes without any bacterial material wereused as a negative control (blank), and freeze-thawed erythrocyte lysatewas used as positive control (100% hemolysate). To determine the % lysisin test samples, plates were read at 405 nm in an ELISA plate reader(HTS 7000 Perkin Elmer), and the reading was expressed as a fraction ofthe positive control.

Real-Time qPCR Assays.

To conduct real-time PCR assays, RNA samples were treated with TurboDNase (Ambion, Austin, Tex., United States) and reverse-transcribedusing M-MLV Reverse Transcriptase (Promega, USA). The real-time PCRassays were performed using the SYBR Green PCR Master Mix Kit (AppliedBiosystems, Foster City, Calif., USA) in a 20-μl reaction volume; allsamples were tested in duplicate. The ribosomal protein S7 gene was usedfor normalization of cDNA templates. Primer sequences used in theseassays are given in Table 1.

TABLE 1 List of gene primers used in gene expression analysesof mosquito tissues post-bacterial challenge. Gene Species Name SequenceAedes aegypti Ribosomal Forward: 5′-GGGACAAATCGGCCAGGCTATC-3′ (SEQ S7ID NO: 4) Reverse: 5′-TCGTGGACGCTTCTGCTTGTTG-3′ (SEQ ID NO: 5)Forward: 5′-TTGTTTGCTTCGTTGCTCTTT-3′ (SEQ ID Defensin-C NO: 6)Reverse: 5′-ATCTCCTACACCGAACCCACT-3′ (SEQ ID NO: 7)Forward: 5′-CCAAGCCTTGTGAACCAGTA-3′ (SEQ ID Cecropin-G NO: 8)Reverse: 5′-GGCCACCTGCTTCAGACT-3′ (SEQ ID NO: 9)Forward: 5′-CGAAGCCGGTGGTCTGAAG-3′ (SEQ ID Cecropin-E NO: 10)Reverse: 5′-ACTACGGGAAGTGCTTTCTCA-3′ (SEQ ID NO: 11) LysozymeForward: 5′-CCACGGCAACTGGATATGTCT-3′ (SEQ ID C NO: 12)Reverse: 5′-TCTGCGTCACCTTGGTGGTAT-3′ (SEQ ID NO: 13) Anopheles PGRP-LCForward: 5′-AGAATACCACACTAAGGCACAGT-3′ gambiae (SEQ ID NO: 14)Reverse: 5′-AGACTTACGATCCTGGTAAATGT-3′ (SEQ ID NO: 15)Forward: 5′-CCAGAGACCAACCAACCACCAA-3′ (SEQ Cecropin 1 ID NO: 16)Reverse: 5′-GCACTGCCAGCACGACAAAGA-3′ (SEQ ID NO: 17)Forward: 5′-CCAAGATGTCGGGCAAGTAT-3′ (SEQ ID FBN9 NO: 18)Reverse: 5′-TTGTGGTACGTCAGCGAGTC-3′ (SEQ ID NO: 19)Forward: 5′-ATGCTCTGCTGTCGTTTGTG-3′ (SEQ ID TEP1 NO: 20)Reverse: 5′-TTCGTGTCCTCCGGTATTTC-3′ (SEQ ID NO: 21)Forward: 5′-TCGGTGAGCAACAGTTTGA-3′ (SEQ ID LRRD7 NO: 22)Reverse: 5′-CTTCATTCCCGCTAATGCT-3′ (SEQ ID NO: 23)Forward: 5′-GCGGTTCCAAAGTTCCGACA-3′ (SEQ ID Defensin 1 NO: 24)Reverse: 5′-AGCGGGACACAAAATTGTTC-3′ (SEQ ID NO: 25)Forward: 5′-CGGAGAAGTCGAAGAAAACG-3′ (SEQ ID Rel2 NO: 26)Reverse: 5′-CACAGGCACACCTGATTGAG-3′ (SEQ ID NO: 27)

Statistical Analysis.

The Mann-Whitney U test, one-way ANOVA with Dunnett's post-test andpairwise Log-Rank tests for survival analysis were conducted using theGraphPad Prism statistical software package (Prism 5.05; GraphPadSoftware, Inc., San Diego, Calif.). Data in FIG. 5 were analyzed usingan ANOVA, followed by a Tukey's test in R (R Foundation for StatisticalComputing).

Results and Discussion

In a previous study, we isolated a Gram-negative bacteriumChromobacterium sp_Panamam (Csp_P) from the midgut of field-collectedAe. aegypti mosquitoes in Panama. The genus Chromobacterium spp.represents soil- and water-associated bacteria of tropical andsubtropical regions, and members of this genus are known to produce avariety of bioactive compounds and to form biofilms. The mostextensively studied member, Chromobacterium violaceum, has been found toproduce violacein, a violet pigment compound with potent antimicrobial,antiparasitic, and tumoricidal activity. Csp_P can be cultured in LuriaBertani (LB) broth and on LB agar, on which it forms flat colonies witha tan color that become darker with time and are opaque when exposed tolight. Csp_P does not produce violacein, but molecular characterizationof its 16s rRNA gene sequence and phylogenetic analysis showed a 98%similarity to Chromobacterium haemolyticum and Chromobacteriumaquaticum, probably its two closest relatives.

Csp_P Colonization of the Mosquito Midgut.

To assess the ability of Csp_P to colonize the mosquito midgut, weexposed antibiotic-treated mosquitoes to a sugar source containing 10⁶colony forming units (CFU)/ml for Ae. aegypti or 10⁸ CFU/ml for An.gambiae for 24 h and then dissected, homogenized and plated the midgutson LB agar plates at 3 days post-exposure. Treatment with antibioticsthrough the sugar meal was performed to remove the native microbialflora which can fluctuate in terms of load and species compositionbetween individual mosquitoes of the same cage and generation, therebycomplicating the interpretation of our data. The presence of the nativemicrobiota would also render it difficult to discriminate the Csp_Pcolonies from those of other species through visual inspection. Csp_Pdisplayed an exceptional ability to rapidly colonize mosquito midguts,showing a prevalence of 80% in An. gambiae and 97% in Ae. aegypti cagepopulations at 3 days after exposure (FIG. 1A). Average bacterial loadsat this time point were approximately 10⁵ and 10⁴ CFU per midgut in Ae.aegypti (FIG. 1B) and An. gambiae (FIG. 1C) females, respectively.

We also tested the ability of Csp_P to colonize the midguts ofnon-antibiotic treated mosquitoes. Because nearly all septic (i.e.,non-antibiotic treated) An. gambiae mosquitoes had died two days afterCsp_P introduction through sugar-feeding at 10⁸ CFU/ml (FIG. 2C), wewere only able to assay prevalence and bacterial load of Csp_P at daysone and two post feeding. At one day after Csp_P ingestion, we foundthat Csp_P was present in all sampled mosquitoes with an averagebacterial load of 5.12×10⁴ (FIG. 1D,E). At two days after Csp_Pexposure, only 5% of Csp_P-fed An. gambiae were still alive (FIG. 2C)and Csp_P was detected in only one (12.5%) of these remaining mosquitoes(FIG. 1D, E). In septic Ae. aegypti mosquitoes that had fed on a 10¹⁰CFU/ml Csp_P-containing sugar solution, we identified Csp_P in 79% ofmosquitoes sampled on day 1 post feeding (FIG. 1F). At three days afterfeeding on the Csp_P containing sugar solution, approximately 30% of theAe. aegypti were still alive (FIG. 2D) and Csp_P was detected in 15% ofthese mosquitoes (FIG. 1F). These data suggest that Csp_P colonized thevast majority of An. gambiae and Ae. aegypti mosquitoes by day 1 postexposure and that Csp_P caused rapid mortality in most individuals. Thesmall percentage that survived up to day 2 or 3, post exposure, may havereceived a small dose of bacteria and succeeded in clearing it by thetime they were dissected. It is difficult to compare the colonizationefficiency between septic and antibiotic treated mosquitoes because thesurvival curves differ dramatically (FIG. 2). While it appears thatCsp_P was better at colonizing the midgut of antibiotic treated An.gambiae (FIG. 1A vs. 1D) and Ae. aegypti (FIG. 1A vs. 1F), ourmeasurement does not take into account that individuals died much morerapidly in the septic population. This rapid mortality likely selectedfor mostly Csp_P negative individuals by day 2 and 3 post-feeding.

Csp_P Exerts Entomopathogenic Activity Upon Mosquito Ingestion andLarval Exposure.

We examined the influence of Csp_P midgut colonization on mosquitolongevity by exposing antibiotic-treated An. gambiae and Ae. aegyptimosquitoes to a sugar source for 24 h containing Csp_P at a finalconcentration of 10⁸ and 10⁶ CFU/ml, respectively, and then monitoringsurvival. This treatment led to a decrease in the longevity of bothspecies when compared to non-exposed control mosquitoes (FIG. 2A, B). Werepeated this experiment with septic (i.e., not antibiotic treated) An.gambiae and Ae. aegypti. We found that feeding on a sugar sourcecontaining Csp_P at a concentration of 10⁸ CFU/ml resulted in rapidmortality of An. gambiae adult females (FIG. 2C). Mortality of septicAe. aegypti females was not increased after feeding on a sugar sourcecontaining Csp_P at a concentration of 10⁶ CFU/ml but was dramaticallyincreased when the sugar meal contained Csp_P at a concentration of 10¹⁰CFU/ml (FIG. 2D). These data suggest that Csp_P has strongentomopathogenic activity regardless of whether other microbes arepresent in the mosquito gut. We observed lower survival in septic An.gambiae and Ae. aegytpi after feeding on a blood meal containing Csp_Pat a final concentration of 10⁸ CFU/ml (FIG. 2E, F). The strongerentomopathogenic effect upon Csp_P introduction through the blood mealwas most likely because the mosquitoes received a large single bacterialdose upon bloodfeeding rather than the multiple low doses that would beexpected during sugar feeding. It is also possible that Csp_Pproliferated to high numbers in the nutritious blood.

To study the influence of Csp_P on larval viability, we placed 2- to4-day-old mosquito larvae in groups of 10 in pools containing 5 mldistilled water supplemented with 50 μl of a 1.0 OD600 liquid culture ofCsp_P, and then monitored survival. This resulted in almost completemortality of An. gambiae and Ae. aegypti larvae over a 3- and 2-dayperiod, respectively, when compared to the control larvae that wereexposed to the normal breeding water microbiota (FIG. 2G, H). Thesestudies suggest that Csp_P-mediated mortality may be the direct resultof a mosquitocidal factor or systemic infection through disseminationinto the hemolymph; alternatively, its colonization of the midgut (orother tissues) might cause mortality indirectly by interfering withvital functions of the mosquito. Studies of Pseudomonas aeruginosacolonization of the Drosophila melanogaster gut have shown that biofilmformation can dramatically affect both dissemination within thehemolymph and fly mortality. Csp_P is capable of forming biofilms invitro, though whether biofilm formation occurs within the mosquitomidgut remains untested. C. violaceum produces cyanide at high celldensity via the cyanide-producing hcnABC operon, a behavior that isreportedly regulated by quorum sensing. Cyanide production by bacteriahas been shown to cause host mortality in both nematodes and insects.Chromobacterium subtsugae has previously been shown to exert oraltoxicity in various insects of agricultural importance, but not in Culexmosquitoes.

Csp_P Colonization of the Mosquito Midgut Compromises PathogenInfection.

To investigate whether the presence of Csp_P in the mosquito midgutcould influence the infection of An. gambiae with P. falciparum and ofAe. aegypti with the dengue virus DENV2, we assayed the infection ofmosquitoes that had been exposed to Csp_P through sugar feeding 2 daysprior to feeding on parasite- or virus-infected blood. Approximately oneweek after An. gambiae had fed on a P. falciparum gametocyte culture,parasite infection was assayed by counting oocyst-stage parasites on thebasal side of the mosquito midgut. DENV2 infection of the midgut of Ae.aegypti was assayed through standard plaque assays 7 days after aninfectious bloodmeal. All experiments were initiated using similarnumbers of adult females for each treatment, but because Csp_P exposurecauses high mortality in adults (FIG. 2), very few Csp_P-fed mosquitoeswere still alive when the parasite and dengue infection assays wereconducted. Nevertheless, we found that surviving mosquitoes exposed toCsp_P through sugar feeding prior to feeding on infectious blooddisplayed significantly increased resistance to P. falciparum infectionand DENV infection (FIG. 3). The inhibition of P. falciparum infectionwas even greater when Csp_P was introduced through thegametocyte-containing blood meal at 10⁶ CFU/ml (FIG. 3B), an effect mostlikely attributable to the larger number of ingested bacteria. Csp_P mayinhibit pathogen infection directly through physical interaction withthe pathogens or the production of anti-pathogen molecules.Alternatively, Csp_P may indirectly inhibit Plasmodium or dengue by (a)altering the long-term physiology or health of the mosquito such thatpathogen infection is inhibited, (b) triggering a mosquito anti-pathogenresponse or (c) selecting for individuals that are more fit to resistCsp_P as well as DENV and Plasmodium infection. However, Csp_P's invitro anti-pathogen activity (discussed below) suggests it has thepotential to directly inhibit pathogen survival in the mosquito gut.Further studies are necessary to elucidate the mechanism by which Csp_Pinhibits the pathogens in vitro and in vivo.

Csp_P Induces Mosquito Innate Immune System Genes.

We have previously shown that the An. gambiae and Ae. aegypti midgutmicrobiota elicit basal immune activity by elevating the expression ofseveral immune factors, including antimicrobial peptides andantipathogen factors. To determine Csp_P's potency in inducing themosquito's innate immune system, we exposed mosquito SUA-5B cells tovarious concentrations of Csp_P and assayed for changes in the activityof a Cecropin1 promoter driving the expression of a luciferase reportergene. We exposed these same cells to Pseudomonas putida, a Gram-negativebacterium that belongs to a bacterial genus commonly found in mosquitomidguts. This experiment showed that Cec1 expression increased withincreasing Csp_P exposure, providing evidence that Csp_P is a potentimmune elicitor (FIG. 4). We also compared the transcript abundance ofmosquito immune genes in midguts from antibiotic-treated naïvemosquitoes to those from mosquitoes that had been provided a sugarsource spiked with Csp_P (10⁸ CFU/ml for An. gambiae and 10⁶ CFU/ml forAe. aegypti) 2 days earlier. We chose to assay gene expression at 2 dayspost exposure because this is the time at which increased mortality dueto infection begins to occur. We hypothesized that infection levels andtherefore any potential immune response would be high at this time. InAe. aegypti, we found that cecropin E and G and defensin C displayed atleast a 2-fold increase in transcript abundance in the midgut of Ae.aegypti colonized with Csp_P bacteria when compared to naïve controls(FIG. 7A). In An. gambiae, we found non-significant trends towardincreased transcript abundance of the Rel2, FBN9 and cecropin genes andtoward decreased transcript abundance of the defensin gene in the midguttissue (FIG. 7B). These data represent a single time pointpost-infection, and while it is possible that additional time points mayreveal dynamic patterns of Csp_P-induced changes in gene expression, ourresults generally agree with the cell culture data, and as a whole showthat Csp_P has an immune-eliciting capacity in the mosquito gut.

Csp_P Inhibits Plasmodium Development and Abolishes Dengue VirusInfectivity In Vitro, Independent of the Mosquito.

To test whether Csp_P could exert a direct anti-Plasmodium oranti-dengue effect in vitro that is independent of the mosquito, weperformed experiments in which parasite development and virusinfectivity were assayed after exposure to various preparations ofeither planktonic or biofilm cultures of Csp_P. Planktonic-state Csp_Pwas obtained by culturing Csp_P in liquid LB at 30° C. overnight on aplatform shaker. Biofilm was produced by culturing Csp_P in LB withoutagitation in a polystyrene 24-well plate at room temperature for 48 h,unless otherwise indicated. The anti-dengue and anti-Plasmodium activityof the following five different preparations of Csp_P was then tested:(a) 1 ml (10⁸ CFU/ml) planktonic-state liquid culture, (b) 1 ml (10⁹CFU/ml) biofilm supernatant consisting of liquid LB drawn off freshlycultured biofilm, (c) 5 mg (10⁹ CFU/ml) fresh biofilm resuspended in1×PBS, (d) 5 mg desiccated biofilm prepared from biofilm collected 1-2days prior to assay and allowed to completely desiccate at roomtemperature and then rehydrated in 1×PBS, (e) 5 mg heat-inactivatedbiofilm prepared by heating biofilm at 90° C. for 24 h, collected 1 dayprior to assay.

Our in vitro assays showed that Csp_P exerts potent anti-Plasmodiumactivity against both asexual and sexual parasite stages. We exposed P.falciparum 3D7 asexual stage parasites to all five bacterialpreparations in vitro. Because bacterial growth can interfere withdetermining parasite number, we removed bacterial cells by filtering allpreparations though a 0.2-μm filter. We found that all filtrates from36-h biofilm preparations (fresh, supernatant, and desiccated) possessedstrong anti-Plasmodium activity, resulting in inhibition of asexualstage parasites at a level comparable to the chloroquine-treatedpositive control (p<0.001, FIG. 5A). We also detected moderateanti-asexual stage activity in planktonic Csp_P preparations (p<0.001),while heat-inactivated Csp_P biofilm and biofilm from another bacterialspecies, Comamonas sp., had no inhibitory effect. We exposed an in vitroPlasmodium ookinete culture to all five filtered bacterial preparationsto assess sexual-stage inhibition and found that the Csp_P 48-h biofilm(fresh, p<0.001; and desiccated, p<0.05) and biofilm supernatant(p<0.001) strongly blocked ookinete development (FIG. 5B). Exposure ofthe ookinete culture to the filtered planktonic Csp_P liquid cultureresulted in a moderate but non-significant inhibition of ookinetedevelopment, and exposure to heat-inactivated Csp_P biofilm or Comomonassp. biofilm filtrate had no effect on ookinete development (FIG. 5B). Wealso tested the effect of Csp_P bacterial preparations on P. falciparumgametocyte viability. Exposure to 42-h fresh biofilm filtrate resultedin 100% inhibition (p<0.001, FIG. 5C) and exposure to 42-h desiccatedbiofilm resulted in approximately 60% inhibition (p<0.05, FIG. 5C) of P.falciparum gametocyte development. Gametocytemia could not be estimatedfor 42-h biofilm supernatant because this preparation caused hemolysisof RBCs (FIG. 5C). However, 36-h biofilm supernatant (which is nothemolytic) caused approximately 60% gametocyte inhibition when comparedto the LB+PBS control (p=0.06, FIG. 8).

To test the inhibitory effect of Csp_P preparations on dengue virusinfectivity in vitro, we mixed dengue virus (10⁶ PFU/ml) in MEM 1:1 witheach of the five bacterial preparations of Csp_P for 45 min. Samplesremained unfiltered during initial exposure to dengue and were filteredthrough a 0.2-μm filter before proceeding with standard plaque assays toavoid contamination of host cells. We found that exposure of denguevirus to a planktonic Csp_P culture did not affect its infectivity inBHK21-15 cells, whereas exposure to Csp_P biofilm, desiccated biofilm,or biofilm supernatant did abolish dengue virus infectivity (p<0.001,FIG. 5D). To better replicate the effect that Csp_P biofilm might haveon dengue virus in human blood, we exposed dengue virus in human bloodto Csp_P fresh biofilm for 45 min. We then filtered theblood+virus/biofilm mixture and assessed dengue virus infectivity bystandard plaque assay. We found that fresh Csp_P biofilm displayedstrong anti-dengue activity when the virus was suspended in human blood(FIG. 5E). Csp_P fresh biofilm was unique in its anti-dengue activity,since the biofilms of several other bacterial isolates from the guts offield-caught mosquitoes did not exert any antiviral activity againstdengue virus in human blood (FIG. 5E). The anti-dengue activity of Csp_Pwas apparently dependent on biofilm maturation, since biofilm grown for24 h showed only weak inhibition when compared to 48-h biofilm (FIG.9A). The Csp_P biofilm-associated anti-Plasmodium and antiviral activitywas also heat-sensitive, since it could be inactivated through a 24-hincubation at 90° C. (FIG. 5A-D).

Bacterial biofilms are composed of a matrix of extracellular polymericsubstances containing polysaccharides, proteins, DNA, and secondarymetabolites. To investigate whether the anti-viral activity could simplybe a result of virus particle sequestration by the biofilm, we mixed adengue virus suspension with biofilm and incubated the mixture for aperiod of 45 min. Samples were then centrifuged, and viral RNA in thesupernatants was quantified by RT-qPCR and compared between experimental(biofilm+DENV) and control (LB+DENV) treatments. Our results indicatedthat the dengue virus was not sequestered by Csp_P biofilm, sincesimilar viral RNA copies were detected in the biofilm-exposed sample andthe LB control sample (FIG. 9B). To investigate whether the loss ofdengue virus infectivity was due to a biofilm-mediated change in the pHof the medium, we measured the pH of a dengue virus-Csp_P biofilmmixture at the end of a 45-min incubation period. The pH measurementsshowed an increase in the pH of the medium from 7.6 to 8.3 (FIG. 10A). Asimilar change in the pH was observed when we used the biofilms of otherbacteria (Pantoea sp. Pasp_P and Proteus sp. Prsp_P) that do not affectdengue virus infectivity (FIG. 10A). To further investigate the effectof pH on dengue virus infectivity, we adjusted the pH of the MEM mediumwith NaOH and HCl to pH values of 5.0, 7.7, 8.5, and 10.0 prior to a45-min incubation with the dengue virus. A decrease in virus infectivitywas only observed after exposure to a pH of 5.0, suggesting that themoderate increase in pH did not mediate the Csp_P biofilm's inhibitionof virus infectivity (FIG. 10B). We also showed that Csp_P biofilm doesnot exert a cytotoxic effect on insect or mammalian cells, as assessedby standard trypan blue cell staining (Invitrogen) (FIG. 11). We finallytested whether the Csp_P biofilm could influence the host cells'susceptibility to dengue virus infection by exposing C6/36 cells toCsp_P biofilm, then removing the biofilm through washes with PBS priorto dengue virus infection assays. This treatment did not influence thevirus's ability to infect the host cells (FIG. 12), suggesting that theanti-DENV activity of Csp_P biofilm is not due to a reduced host cellsusceptibility to the virus but is likely a direct anti-viral effect.

Csp_P Produces Broad-Spectrum Antibacterial Activity(ies).

To provide baseline information on the potential production ofantibacterial factors by Csp_P, we performed a basic growth inhibitionassay by investigating the ability of a number of other mosquitomidgut-derived bacterial isolates (Ae. aegypti-derived microbiota:Ps.sp=Presudomonas sp., Pr.sp=Proteus sp., Cs.p_P=C.sp_P, C.viol=C.violaceum, Pa.sp=Paenobacillus sp.; An. gambiae-derived microbiota:Co.sp=Comamonas sp., Ac.sp=Acinetobacter sp., Ps.pu=Pseudomonas putida,En.sp=Enterobacter sp., Pn.sp=Pantoea sp., Ps.sp=Pseudomonas sp.,S.sp=Serratia sp., Ch.sp=Chryseobacterium sp.) to grow in proximity toCsp_P on LB agar plates (FIG. 6). Csp_P was streaked on LB agar andallowed to grow for 48 hours. Midgut-derived bacterial isolates werethen vertically streaked up to the Csp_P streak, and allowed to grow inthe presence of Csp_P. This assay showed a prominent growth inhibitionzone around the Csp_P streak, with inhibition of the growth of all thebacterial isolates that were derived from field-collected Ae. aegyptiand An. gambiae, including a close relative known for its production ofa variety of bioactive factors, C. violaceum (FIG. 6A).

CONCLUSION

Insect-bacteria associations can influence vector competence in multipleways; these include shortening the insect's life span, blockinginfection with human pathogens by the production of bioactiveanti-pathogen factors, and eliciting the insect immune system. We haveidentified a Chromobacterium sp_Panamam (Csp_P) bacterium from themidgut of field-derived Aedes aegypti that exerts broad-spectrumanti-pathogen activity against Plasmodium and dengue virus.Specifically, Csp_P renders An. gambiae and Ae. aegypti more resistantto infection by the human malaria parasite Plasmodium falciparum anddengue virus, respectively. Csp_P inhibits the growth of a variety ofother bacterial species found in the mosquito midgut and is capable ofrapidly colonizing the mosquito midgut. Csp_P appears to exertentomopathogenic activity, since exposure of larvae to Csp_P in thebreeding water and ingestion of Csp_P by adult mosquitoes result in highmosquito mortality. It is possible that Csp_P could be effectively usedas a transmission blocking agent if it was delivered to mosquitoesthrough baited sugar traps. Csp_P's ability to colonize the mosquito gutcould be further enhanced through established selection procedures basedon consecutive passages of the bacterium through the mosquito intestine.Csp_P could be used alone in baited sugar traps or in combination withother microbes that have also been shown to either kill the mosquito orreduce pathogen infection, or both, when present in the mosquito gut.The larvicidal activity of Csp_P also renders it interesting forpotential use in mosquito population suppression. The anti-pathogenactivities of Csp_P appear to be mediated by bacteria-producedmetabolites that also inhibit parasite and virus infection in vitro,making them interesting as possible lead compounds for transmissionblocking and therapeutic drug development. The entomopathogenic,anti-bacterial, anti-viral, and anti-Plasmodium properties of Csp_P makethis bacterium a particularly interesting candidate for the developmentof novel control strategies for the two most important vector-bornediseases, and they therefore warrant further in-depth study.

Example 2: Live Chromobacterium Csp_P is a General Mosquitocidal, andMalaria and Dengue Transmission-Blocking Agent

Live Chromobacterium Csp_P can be easily exposed to adult mosquitoesthrough artificial sugar feeding and to larvae in the breeding water.Devices (sugar-bait stations) to expose mosquitoes to toxic agents havebeen developed and validated/used for mosquito control (FIG. 14).Exposure of adults to toxic agents through ingestion can also beachieved through direct spraying on the habitat, as in the case ofTerminix's Attractive Targeted Sugar Bait which is widely used formosquito control in the USA. Csp_P readily colonizes the gut or a largeproportion of mosquitoes in a cage population when provided throughartificial nectar feeding (FIG. 15A). Exposure of larvae or adultmosquitoes to Csp_P results in a significant killing (FIG. 15B). Thiskilling activity has been validated for the malaria vectors Anophelesgambiae (African vector), Anopheles stephensi (Asian vector) andAnopheles albimanus (South American vector), the dengue vectors Aedesaegypti (worldwide vector) and Aedes albopictus (worldwide vector), andthe West Nile Virus vector Culex pipiens (world-wide vector) (data notshown). Furthermore, the presence of Csp_P in adult Anopheles and Aedesgut tissue blocks Plasmodium and dengue virus infection, respectively(FIG. 15C). Hence, exposure of larvae and adult mosquitoes to Csp_Pattenuates the transmission of disease by killing the vector and byblocking the pathogen in the vector (FIGS. 15B and 15C). The combinedlarvicidal and adulticidal activities of Csp_P is more potent than thatof other studied Chromobacterium sp strains, including Cromobacteriumsubtsugae which is the active component of the agricultural biopesticideGrandevo which is marketed by Marrone Bio Innovations (FIG. 15D).

Secreted Chromobacterium Csp_P Metabolites have Mosquitocidal Activity.

Exposure of adult mosquitoes to a Csp_P bacteria-free biofilm culturesupernatant through artificial nectar feeding results in significantkilling, indicating the presence of mosquitocidal agents in the filtratepreparation (FIG. 16A). Csp_P likely secrets stable metabolites withmosquitocidal activity. Reverse Phase Liquid Chromatography (RPLC) ofthe Csp_P bacteria-free biofilm culture supernatant identifies severalmetabolite peaks that may contain one or more Csp_P biofilm culturesecreted metabolites (FIG. 16B). Mosquitocidal assays with pools of RPLCfractions that contain metabolite peaks identified one fraction poolthat kill adult mosquitoes upon ingestion through an artificial nectar(FIG. 16C). This fraction pool likely contains the mosquitocidalmetabolite and is subjected to further fractionation and bioassays todefine the active component(s).

The versatility of Csp_P mosquitocidal activities that can target bothlarval and adult stages is unique, in contrast to the widely usedmosquitocidal biopesticides which are based on live Bacillusthuringiensis and Bacillus sphaericus bacteria that only act against thelarval stages. Mosquito resistance has been observed for both Bacillusthuringiensis and Bacillus sphaericus. The pathogen-blocking activity ofCsp_P in the mosquito gut further potentiates its utility formosquito-based malaria and dengue transmission blocking (FIG. 14).

We claim:
 1. A method for controlling malaria and dengue virustransmission via mosquitoes comprising applying in an area where themosquitoes are to be controlled a composition comprising mosquito nectarfeed and Chromobacterium sp_Panamam (Csp_P).
 2. The method of claim 1,wherein the mosquitoes comprise Anopheles and Aedes mosquitoes.
 3. Themethod of claim 2, wherein the Anopheles mosquitoes comprise Anophelesgambiae mosquitoes.
 4. The method of claim 2, wherein the Aedesmosquitoes comprise Aedes aegypti mosquitoes.
 5. A method forcontrolling Anopheles and Aedes mosquitoes comprising applying in anarea where the mosquitoes are to be controlled a composition comprisingan effective insect control amount of a supernatant, filtrate or extractof a biologically pure culture of Csp_P.
 6. The method of claim 5,wherein the composition further comprise a sugar source.
 7. The methodof claim 6, wherein the sugar comprises sucrose, dextrose and/orfructose.
 8. The method of claim 1, wherein the Csp_P is the bacteriahaving the characteristics of ATCC Designation No. PTA-121570.
 9. Themethod of claim 5, wherein the Csp_P is the bacteria having thecharacteristics of ATCC Designation No. PTA-121570.
 10. The method ofclaim 1, wherein the Csp-P comprises a biofilm.
 11. The method of claim10, wherein the biofilm is fresh or desiccated.
 12. The method of claim1, wherein the Csp-P comprises a culture.
 13. The method of claim 1,wherein the Csp_P comprises a supernatant.
 14. The method of claim 1,wherein the Csp_P comprises a filtrate.
 15. The method of claim 1,wherein the Csp_P has the 16s rDNA gene sequence of SEQ ID NO:1.
 16. Themethod of claim 1, wherein the nectar feed comprises one or more ofsucrose, dextrose and fructose.